My long-term career goal is to carry out research that is focused on the molecular basis of disease. My immediate goal is to advance my career by starting to build an independent research program under the guidance of two distinctive and well-established mentors. Dr. Dawson will provide supervision in all aspects of the career development plan and Dr. Larsson will contribute in designing and interpreting experiments on state-dependence of cysteine accessibility. The long-range goal of the proposed research is to determine if there are structural components in the transmembrane domains (TMDs) of CFTR channel that are involved in the conformational changes associated with channel gating. It is well established that channel gating is controlled by the intracellular domains, but results from several studies have suggested that the pore domain is an important part of the gating mechanism. Little is known, however, about how the binding and hydrolysis of ATP in the nucleotide binding domains are translated into either the movement or a conformational change in TMDs that opens or closes the channel pore. The proposed research will focus on three specific aims: 1) To identify residues exhibiting state-dependent accessibility to Au(CN)2-. 2) To investigate the effect of Au(CN)2- on the single-channel function in constructs exhibiting state-dependent accessibility to Au(CN)2-. 3) To investigate the effects of other thiol-directed reagents on the function of constructs that show state-dependent accessibility to Au(CN)2-. The working hypothesis that will guide the proposed research is that CFTR shares the fundamental functional features of the ATP Binding Cassette (ABC) family in which conformational changes in the TMDs occur during the transport process. The research strategy is to assay the effects of thiol-directed reagents on engineered cysteines in the TMD's of CFTR using inside-out or outside-out patches that will allow the access to both the intracellular and the extracellular solution.